Intimal thickening inhibitory agent

ABSTRACT

An intimal thickening inhibitory agent comprising, as an active ingredient, a compound represented by formula (1):                    
     wherein x represents an oxygen atom or a group of formula (2) 
     
       
         —S—(O) n   (2) 
       
     
     wherein n represents an integer of from 0 to 2, R 1  represents a hydrogen atom or an acyl group; R 2  represents a hydrogen atom, a lower alkyl group or a lower alkenyl group; R 3  represents a lower alkyl group; and R 4 , R 5 , and R 6 , which may be the same or different, each represent a hydrogen atom or a substituted or unsubstituted alkyl group; or R 3  and R 4  may be taken together to form a 5-membered ring; or R 5  and R 6  may be taken together to form a cycloalkyl group; provided that R 6  is nil when R 3  and R 4  are taken together to form benzofuran or benzo[b]thiophene.

This is a continuation of patent application Ser. No. 09/068,610, nowU.S. Pat. No. 6,013,353, which was nationalized May 6, 1998, theinternational application PCT/JP96/03279 of which was filed Nov. 8,1996.

FIELD OF THE INVENTION

This invention relates to an intimal thickening inhibitory agent andmore particularly an intimal thickening inhibitory agent comprising a2,6-di-t-butylphenol derivative as an active ingredient.

BACKGROUND OF THE INVENTION

It is known that coronary sclerosis is a primary cause of ischemic heartdiseases such as angina pectoris and cardiac infarction. Narrowing ofthe vascular lumen resulting from arteriosclerotic thickening of theintima brings about nutrition and oxygen deficiencies in the myocardialtissues to induce the above diseases. Percutaneous transluminal coronaryangioplasty (hereinafter abbreviated as “PTCA”) that has recently beendeveloped as a treatment for the ischemic heart diseases such as anginapectoris and cardiac infarction is to physically dilate a blood vesselby inflating a balloon at the stenosis region of the coronary artery.However, the problem which has been recognized from the beginning ofdevelopment of this treatment is that restenosis appears at the treatedregion within 3 to 6 months after the angioplasty at a frequency ofabout 40% (see Circulation, 77, pp. 361-371 (1988)).

Up to the present time, use of anticoagulants, antiplatelet agents ordrugs having an inhibitory effect on proliferation of vascular smoothmuscle cells has been attempted to prevent stenosis due toarteriosclerotic intimal thickening or restenosis after PTCA. Thus, anextensive research for such drug has been conducted (see, for example,JP 2-121922 A/90, JP 3-83923 A/91, JP 3-118383 A/91, JP 4-99775 A/92, JP4-154720 A/92, JP 6-135829 A/94, JP 6-206842 A/94, JP 7-25768 A/95, JP7-149641 A/95 and JP 7-223958 A/95). However, there has been found nodrug having clinically sufficient inhibitory effect on vascular stenosisdue to arteriosclerotic intimal thickening or restenosis due to intimalthickening after PTCA (see, Nihon Rinsvo, 52 (extra ed.), pp. 869-872(1994)).

DISCLOSURE OF THE INVENTION

As a result of an extensive research in an attempt to solve theforegoing problem, it has been found that a compound represented byformula (1):

wherein X represents an oxygen atom or a group of formula (2);

—S—(O)_(n)  (2)

wherein n represents an integer of from 0 to 2;

R₁ represents a hydrogen atom or an acyl group;

R₂ represents a hydrogen atom, a lower alkyl group or a lower alkenylgroup;

R₃ represents a lower alkyl group; and

R₄, R₅, and R₆, which may be the same or different, each represent ahydrogen atom, a substituted or unsubstituted alkyl group, a substitutedor unsubstituted alkenyl group, a substituted or unsubstituted alkynylgroup, or a substituted or unsubstituted aryl group; or

R₃ and R₄ may be taken together to form a 5-membered ring; or

R₅ and R₆ may be taken together to form a cycloalkyl group or aheterocyclic ring derived from a cycloalkyl group by substituting anyone or more methylene groups on the ring with oxygen atoms, sulfur atomsor alkyl-substituted nitrogen atoms;

provided that R₆ is nil when R₃ and R₄ are taken together to form abenzofuran ring, a benzo[b]thiophene ring, a benzo[b]thiophene-1-oxidering or a benzo[b]thiophene-1,1-dioxide ring,

exhibits an excellent inhibitory effect on proliferation of vascularsmooth muscle cells as well as on intimal thickening in balloon injurymodels.

The fact that the compound represented by formula (1) is effective inthe treatment and prevention of ischemic organopathy such asarteriosclerosis, cardiac infarction and apoplexy has already beenrevealed (see JP 6-206842 A/94, WO 94-08930, and WO 95-27710).

BEST MODE FOR CARRYING OUT THE INVENTION

In the above formula (1), the acyl groups include an acetyl group, aformyl group, a propionyl group, a benzoyl group, a benzyloxycarbonylgroup, an aminoacetyl group, an N-methylaminoacetyl group, and anN,N-dimethylaminoacetyl group. The term “lower alkyl group” means astraight- or branched-chain alkyl group having 1 to 6 carbon atoms, forexample, a methyl group, an ethyl group, a n-propyl group, an i-propylgroup, a n-butyl group, a sec-butyl group, and a tert-butyl group. Theterm “lower alkenyl group” means a straight- or branched-chain alkenylgroup having 2 to 6 carbon atoms, for example, a vinyl group, an allylgroup, a butenyl group, and a pentenyl group.

The alkyl group represented by R₄, R₅, or R₆ is a straight- orbranched-chain alkyl group having 1 to 20 carbon atoms, for example, amethyl group, an ethyl group, a n-propyl group, an i-propyl group, an-butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, ahexyl group, a heptyl group, an octyl group, a nonyl group, and a decylgroup. The alkenyl group is a straight- or branched-chain alkenyl grouphaving 2 to 20 carbon atoms, for example, a vinyl group, an allyl group,a butenyl group, a pentenyl group, a geranyl group, and a farnesylgroup. The alkynyl group is a straight- or branched-chain alkynyl grouphaving 2 to 20 carbon atoms, for example, an ethynyl group, a propynylgroup, and a butynyl group. The aryl group is a monovalent substituentderived from an aromatic hydrocarbon by removing one hydrogen atom, forexample, a phenyl group, a tolyl group, a xylyl group, a biphenyl group,a naphthyl group, an anthryl group, and a phenanthryl group.Substituents in the substituted alkyl, alkenyl, alkynyl or aryl groupinclude a halogen atom, a lower alkyl group, a hydroxyl group, an aminogroup, an alkoxy group, an aryloxy group, a nitro group, and atrifluoromethyl group.

The 5-membered rings formed by R₃ and R₄ include a furan ring, adihydrofuran ring, a thiophene ring, and a dihydrothiophene ring, whichare taken together with the benzene ring to form a benzofuran ring, adihydrobenzofuran ring, a benzo[b]thiophene ring, and adihydrobenzothiophene ring, respectively.

The cycloalkyl group is a cycloalkyl group having 3 to 8 carbon atoms,for example, a cyclopropyl group, a cyclobutyl group, a cyclopentylgroup, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.The heterocyclic rings derived from the cycloalkyl group by substitutingany one or more methylene groups on the ring with oxygen atoms, sulfuratoms or alkyl-substituted nitrogen atoms include, for example, atetrahydropyranyl group.

Where X in formula (1) is an oxygen atom,

R₁ is preferably a hydrogen atom, an acetyl group, a benzyloxycarbonylgroup, an aminoacetyl group, an N-methylaminoacetyl group or anN,N-dimethylaminoacetyl group, particularly a hydrogen atom, an acetylgroup or an N,N-dimethylaminoacetyl group;

R₂ is preferably a hydrogen atom, a methyl group or a n-propyl group,particularly a hydrogen atom;

R₃ and R₄ are preferably taken together to form a furan ring or adihydrofuran ring, particularly a dihydrofuran ring;

R₅ is preferably a hydrogen atom, a methyl group, a n-butyl group, an-pentyl group, a n-hexyl group or an i-amyl group, particularly an-pentyl group; and

R₆ is preferably a hydrogen atom, a methyl group, a n-butyl group, an-pentyl group, a n-hexyl group or an i-amyl group, particularly an-pentyl group; or

R₅ and R₆ are preferably taken together to form a cyclopentyl group, acyclohexyl group, a cycloheptyl group, a cyclooctyl group, or atetrahydropyranyl group, particularly a cyclohexyl group, a cycloheptylgroup, or a cyclooctyl group.

Where X in formula (1) is a group represented by formula (2):

—S—(O)_(n)  (2)

wherein n is an integer of from 0 to 2,

R₁ is preferably a hydrogen atom, an acetyl group, a benzyloxycarbonylgroup, an aminoacetyl group, an N-methylaminoacetyl group or anN,N-dimethylaminoacetyl group, particularly a hydrogen atom, an acetylgroup or an N,N-dimethylaminoacetyl group;

R₂ is preferably a hydrogen atom, a methyl group or an n-propyl group,particularly a hydrogen atom;

R₃ and R₄ are preferably taken together to form a thiophene ring or adihydrothiophene ring, particularly a dihydrothiophene ring;

R₅ is preferably a hydrogen atom, a methyl group, a n-butyl group, an-pentyl group, a n-hexyl group or an i-amyl group, particularly an-pentyl group; and

R₆ is preferably a hydrogen atom, a methyl group, a n-butyl group, an-pentyl group, a n-hexyl group or an i-amyl group, particularly an-pentyl group; or

R₅ and R₆ are preferably taken together to form a cyclopentyl group, acyclohexyl group, a cycloheptyl group, a cyclooctyl group, or atetrahydropyranyl group, particularly a cyclohexyl group, a cycloheptylgroup, or a cyclooctyl group; and

n is preferably 0 or 1, particularly 0.

Examples of the specific compounds used for the inhibitory agent of thepresent invention include4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2-methyl-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-diethyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-di-n-propyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-d i-n-butyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2-octylbenzofuran,4,6-di-t-butyl-5-hydroxy-2-octyl-2,3-dihydrobenzofuran,2,4,6-tri-t-butyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-di-i-propyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-diphenyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-dibenzyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2-chloromethyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cyclopentane,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cyclohexane,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cycloheptane,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cyclooctane,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-4′-tetrahydropyran,5-hydroxy-4,6-di-t-butyl-2,2-dimethyl-7-propyl-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxybenzofuran,4,6-di-t-butyl-5-hydroxy-2-methylbenzofuran,2,4,6-tri-t-butyl-5-hydroxybenzofuran,2,6-di-t-butyl-3-methyl-4-propyloxyphenol,4-allyloxy-2,6-di-t-butyl-3-methylphenol,1,3-bis(3,5-di-t-butyl-4-hydroxy-2-methylphenoxy)propane,4,6-di-t-butyl-2,2-di-n-pentyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-di-n-octyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-di-n-heptyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-2,2-di-n-hexyl-5-hydroxy-2,3-dihydrobenzofuran,2,2-di-i-amyl-4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2-methyl-2-(4,8,12-trimethyl-trideca-3(E),7(E),11-trienyl)-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2-methyl-2-(4′,8′,12′-trimethyltridecyl)-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2-(5-hydroxy-4-methyl-3(E)-pentenyl)-2-methyl-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2-hydroxymethyl-2-methyl-2,3-dihydrobenzofuran,4,6-di-t-butyl-5-hydroxy-2,2-di-n-pentyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2-methyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-dimethyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxybenzo[b]thiophene,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-diethyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-di-n-propyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-di-i-propyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-di-n-butyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-di-i-amyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-di-n-hexyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-di-n-heptyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-di-n-octyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-diphenyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-dibenzyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2-methyl-2-(4,8,12-trimethyltrideca-3(E),7(E),11-trienyl)-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2-methyl-2-(4,8,12-trimethyltridecyl)-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2-n-octyl-2,3-dihydrobenzothiophene,2,4,6-tri-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,2-dimethyl-7-n-propyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene-2-spiro-1′-cyclopentane,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene-2-spiro-1′-cyclohexane,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene-2-spiro-1′-cycloheptane,4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene-2-spiro-1′-cyclooctane,4,6-di-t-butyl-2-methyl-5-hydroxybenzo[b]thiophene,2,4,6-tri-t-butyl-5-hydroxybenzo[b]thiophene,4,6-di-t-butyl-2-octyl-5-hydroxybenzo[b]thiophene,4,6-di-t-butyl-5-hydroxy-2-(N,N-dimethylaminomethyl)-2-methyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2-hydroxymethyl-2-methyl-2,3-dihydrobenzothiophene,4,6-di-t-butyl-5-hydroxy-2-methyl-2-(4,8-dimethyl-nona-3(E),7-dienyl)-2,3-dihydrobenzothiophene,and4,6-di-t-butyl-5-hydroxy-2-methyl-2-(4,8-dimethylnonyl)-2,3-dihydrobenzothiophene.

The compound represented by formula (1) used in the present invention issynthesized in accordance with, for example, the process described in JP6-206842 A/94 or WO 95-27710.

The vascular intimal thickening inhibitory agent of the presentinvention can be used in the form of a pharmaceutical compositioncomprising a compound of formula (1) together with physiologicallynon-toxic solid or liquid pharmaceutical carrier. The pharmaceuticalcomposition may be in a variety of dosage forms depending on theadministration route. Suitable dosage forms include tablets, granules,pills, capsules, solutions, syrups, suspensions, emulsions, andinjection solutions. Usual pharmaceutical carriers, such as excipient,binder, disintegrant, lubricant, coating agent, solubilizer, emulsifier,suspending agent, stabilizer, and solvents, may be used.

The compound represented by formula (1) and the above pharmaceuticalcomposition according to the present invention can be administered by anoral or parenteral route such as intravenous injection or as a sustainedrelease formulation or by a topical route, such as through a catheter.

The actual dosage of the compound of formula (1) to be required forinhibiting restenosis after PTCA depends on the age of the patient, theseverity of the condition to be treated, the administration route, andthe like. However, the effective dosage which is generally accepted willbe in the range of, for example, 1 to 1000 mg, preferably 100 to 300 mgper day in the treatment of adult human. The dosage is preferably givenin 1 to 3 dose administrations daily to the patient in need of suchtreatment.

EXAMPLES

The present invention will now be illustrated with reference to thefollowing Examples, but it should be understood that the presentinvention is not construed as being limited thereto.

The compounds of Examples 1 to 46 were synthesized in accordance withthe process described in JP 6-206842 A/94.

Example 1 4,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran Example 24,6-Di-t-butyl-5-hydroxy-2-methyl-2,3-dihydrobenzofuran Example 35-Acetoxy-4,6-di-t-butyl-2,2-dimethyl-2,3-dihydrobenzofuran Example 44,6-Di-t-butyl-5-hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran Example 55-Acetoxy-4,6-di-t-butyl-2,2-diethyl-2,3-dihydrobenzofuran Example 64,6-Di-t-butyl-2,2-diethyl-5-hydroxy-2,3-dihydrobenzofuran Example 74,6-Di-t-butyl-2,2-di-n-propyl-5-hydroxy-2,3-dihydrobenzofuran Example 84,6-Di-t-butyl-2,2-di-n-butyl-5-hydroxy-2,3-dihydrobenzofuran Example 95-Acetoxy-4,6-di-t-butyl-2-(1-octenyl)benzofuran Example 105-Acetoxy-4,6-di-t-butyl-2-octylbenzofuran Example 114,6-Di-t-butyl-5-hydroxy-2-octylbenzofuran Example 124,6-Di-t-butyl-5-hydroxy-2-octyl-2,3-dihydrobenzofuran Example 132,4,6-Tri-t-butyl-5-hydroxy-2,3-dihydrobenzofuran Example 144,6-Di-t-butyl-2,2-di-i-propyl-5-hydroxy-2,3-dihydrobenzofuran Example15 4,6-Di-t-butyl-2,2-diphenyl-5-hydroxy-2,3-dihydrobenzofuran Example16 4,6-Di-t-butyl-2,2-dibenzyl-5-hydroxy-2,3-dihydrobenzofuran Example17 4,6-Di-t-butyl-2-chloromethyl-5-hydroxy-2,3-dihydrobenzofuran Example184,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cyclopentaneExample 194,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cyclohexaneExample 204,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cycloheptaneExample 214,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1′-cyclooctaneExample 224,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-4′-tetrahydropyranExample 234-Acetoxy-3,5-di-t-butyl-1-(2-methyl-2-propenyloxy)-2-propylbenzeneExample 245-Acetoxy-4,6-di-t-butyl-2,2-dimethyl-7-propyl-2,3-dihydrobenzofuranExample 255-Hydroxy-4,6-di-t-butyl-2,2-dimethyl-7-propyl-2,3-dihydrobenzofuranExample 26 5-Acetoxy-4,6-di-t-butylbenzofuran Example 274,6-Di-t-butyl-5-hydroxybenzofuran Example 284,6-Di-t-butyl-5-hydroxy-2-methylbenzofuran Example 292,4,6-Tri-t-butyl-5-hydroxybenzofuran Example 301-Acetoxy-2,6-di-t-butyl-3-methyl-4-propyloxybenzene Example 312,6-Di-t-butyl-3-methyl-4-propyloxyphenol Example 321-Acetoxy-4-allyloxy-2,6-di-t-butyl-3-methylbenzene Example 334-Allyloxy-2,6-di-t-butyl-3-methylphenol Example 341,3-Bis(4-acetoxy-3,5-di-t-butyl-2-methylphenoxy)propane Example 351,3-Bis(3,5-di-t-butyl-4-hydroxy-2-methylphenoxy)propane Example 364,6-Di-t-butyl-2,2-di-n-pentyl-5-hydroxy-2,3-dihydrobenzofuran Example37 4,6-Di-t-butyl-2,2-di-n-octyl-5-hydroxy-2,3-dihydrobenzofuran Example38 4,6-Di-t-butyl-2,2-di-n-heptyl-5-hydroxy-2,3-dihydrobenzofuranExample 39 4,6-Di-t-butyl-2,2-di-n-hexyl-5-hydroxy-2,3-dihydrobenzofuranExample 40 5-Acetoxy-2,2-di-i-amyl-4,6-di-t-butyl-2,3-dihydrobenzofuranExample 41 2,2-Di-i-amyl-4,6-di-t-butyl-5-hydroxy-2,3-dihydrobenzofuranExample 425-Acetoxy-4,6-di-t-butyl-2-methyl-2-(4,8,12-trimethyl-trideca-3(E),7(E),11-trienyl)-2,3-dihydrobenzofuran Example 434,6-Di-t-butyl-5-hydroxy-2-methyl-2-(4,8,12-trimethyl-trideca-3(E),7(E),11-trienyl)-2,3-dihydrobenzofuranExample 444,6-Di-t-butyl-5-hydroxy-2-methyl-2-(4′,8′,12′-trimethyltridecyl)-2,3-dihydrobenzofuranExample 455-Acetoxy-4,6-di-t-butyl-2-(5-hydroxy-4-methyl-3(E)-pentenyl)-2-methyl-2,3-dihydrobenzofuranExample 46 4,6-Di-t-butyl-5-hydroxy-2-(5-hydroxy-4-methyl-3(E)-pentenyl) -2-methyl-2,3-dihydrobenzofuran Example 475-Acetoxy-4,6-di-t-butyl-2-hydroxymethyl-2-methyl-2,3-dihydrobenzofuran

In 200 ml of chloroform was dissolved 10.0 g of4-acetoxy-3,5-di-t-butyl-2-(2-methyl-2-propenyl)phenol synthesized inaccordance with JP 6-206842 A/94, and 11.0 g of m-chloroperbenzoic acidwas added thereto, followed by heating under reflux for one day. Aftercooling, a saturated aqueous solution of sodium thiosulfate was added tothe reaction mixture, and the mixture was extracted with chloroform. Theorganic layer was washed with a saturated sodium chloride aqueoussolution, dried over anhydrous magnesium sulfate, and concentrated. Theresidue was purified by silica gel column chromatography (25% ethylacetate in n-hexane) to give 7.3 g (yield: 70%) of5-acetoxy-4,6-di-t-butyl-2-hydroxymethyl-2-methyl-2,3-dihydrobenzofuran(rotational isomer mixture) as a colorless oil.

¹H-NMR (270 MHz, CDCl₃) δ ppm: 1.30 (s, 9H), 1.37 (s, 9H), 1.38 (s,1.5H), 1.45 (s, 1.5H), 2.30 (s, 3H), 3.06 (d, 0.5H, J=15.5 Hz), 3.16 (d,0.5H, J=15.5 Hz), 3.38 (d, 0.5H, J=15.5 Hz), 3.52 (d, 0.5H, J=15.5 Hz),3.58—3.72 (m, 2H), 6.75 (s, 0.5H), 6.76 (s, 0.5H). Mass: 334 (M⁺).

Example 484,6-Di-t-butyl-5-hydroxy-2-hydroxymethyl-2-methyl-2,3-dihydrobenzofuran

A solution of 500 mg of5-acetoxy-4,6-di-t-butyl-2-hydroxymethyl-2-methyl-2,3-dihydrobenzofuranin 7 ml of tetrahydrofuran was added dropwise to a suspension of 114 mgof lithium aluminum hydride in 3 ml of tetrahydrofuran under nitrogenatmosphere. The reaction mixture was heated under reflux for 2 hours.After allowing the mixture to attain room temperature, ethyl acetate wasadded dropwise, 10% hydrochloric acid was added thereto, and the mixturewas extracted with ethyl acetate. The organic layer was dried overanhydrous magnesium sulfate and concentrated. The residue was purifiedby silica gel column chromatography (20% ethyl acetate in n-hexane) toyield 320 mg (73%) of4,6-di-t-butyl-5-hydroxy-2-hydroxymethyl-2-methyl-2,3-dihydrobenzofuranas a white solid.

Melting point: 126-128° C.; ¹H-NMR (270 MHz, CDCl₃) δ ppm: 1.38 (s, 3H),1.40 (s, 9H), 1.49 (s, 9H), 2.04 (bs, 1H), 3.14 (d, 1H, J=15.5 Hz), 3.45(d, 1H, J=15.5 Hz), 3.59 (d, 2H, J=1.65 Hz), 4.74 (s, 1H), 6.65 (s, 1H);IR (cm⁻¹): 3648, 3448, 2960; Mass: 292 (M⁺).

The compounds of Examples 49 to 67 were synthesized according to theprocess described in WO 95-27710.

Example 495-Acetoxy-4,6-di-t-butyl-2,2-di-n-pentyl-2,3-dihydrobenzothiopheneExample 504,6-Di-t-butyl-5-hydroxy-2,2-di-n-pentyl-2,3-dihydrobenzothiopheneExample 51 4,6-Di-t-butyl-5-hydroxy-2-methyl-2,3-dihydrobenzothiopheneExample 524,6-Di-t-butyl-5-hydroxy-2,2-dimethyl-2,3-dihydrobenzothiophene Example53 5-Acetoxy-4,6-di-t-butylbenzo[b]thiophene Example 544,6-Di-t-butyl-5-hydroxybenzo[b]thiophene Example 555-Acetoxy-4,6-di-t-butyldioxobenzo[b]thiophene-1,1-dioxide Example 565-Acetoxy-4,6-di-t-butyl-2,3-dihydrobenzothiophene-1,1-dioxide Example57 4,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene Example 584,6-Di-t-butyl-5-hydroxy-2,3-dihydrobenzothiophene-2-spiro-1′-cyclohexaneExample 595-Acetoxy-4,6-di-t-butyl-2-iodomethyl-2-methyl-2,3-dihydrobenzothiopheneExample 605-Acetoxy-4,6-di-t-butyl-2-(N,N-dimethylaminomethyl)-2-methyl-2,3-dihydrobenzothiopheneExample 614,6-Di-t-butyl-5-hydroxy-2-(N,N-dimethylaminomethyl)-2-methyl-2,3-dihydrobenzothiopheneExample 625-Acetoxy-2-acetoxymethyl-4,6-di-t-butyl-2-methyl-2,3-dihydrobenzothiopheneExample 634,6-Di-t-butyl-5-hydroxy-2-hydroxymethyl-2-methyl-2,3-dihydrobenzothiopheneExample 64 4,6-Di-t-butyl-5-hydroxy-2-methyl-2-(4,8-dimethyl-nona-3(E),7-dienyl)-2,3-dihydrobenzothiophene Example 654,6-Di-t-butyl-5-hydroxy-2-methyl-2-(4,8-dimethylnonyl)-2,3-dihydrobenzothiopheneExample 664,6-Di-t-butyl-5-hydroxy-2-methyl-2-(4,8,12-trimethyl-trideca-3(E),7(E),11-trienyl)-2,3-dihydrobenzothiophene Example 674,6-Di-t-butyl-5-hydroxy-2-methyl-2-(4,8,12-trimethyltridecyl)-2,3-dihydrobenzothiopheneTest Example 1 Inhibitory Effect on Proliferation of Vascular SmoothMuscle Cells (in vitro)

In order to assess the inhibitory effect of a compound of the presentinvention on serum-stimulated pro-liferation of vascular smooth musclecells in vitro, the compound obtained in Example 36 was tested usingA7r5 cells derived from the aorta of a rat (ATCC-CRL-1444).

Using DMEM medium containing 10% FBS, the cells were placed on 96-wellmicrotiter plates with 5×10³ cells/0.2 ml/well and then incubated. Themedium was exchanged for 2% FBS-containing DMEM medium on the third dayand then for 0.2% FBS-containing DMEM medium on the fourth day from thestart of the incubation. After 48-hour incubation in the 0.2%FBS-containing DMEM medium, the medium was removed, 0.1 ml of DMEMmedium and the test compound were added, and the cells were stimulatedwith serum (2% FBS) to proliferate. The test compound was added to thewells in the form of a solution or suspension in ethanol in an amount of0.5 μl/well to final compound concentrations indicated in Table 1 belowand the resultant ethanol concentration of 0.5% in each well. BrdU waspulsed to the wells from the 16th to 38th hours or from the 38th to 64thhours after the stimulation to a final concentration of 10 μM, and theuptake of BrdU into DNA was evaluated as an indication of cellproliferation. The BrdU uptake was measured by means of an EIA kitcontaining an anti-BrdU antibody (5-Bromo-2′-deoxy-uridine Labeling andDetection Kit III, available from Bohringer Mannheim Biochemica). Theresults obtained are shown in Table 1 below.

TABLE 1 Inhibitory Effect on Proliferation of Vascular Smooth MuscleCells (1) Absorbance (difference Concn between 405 nm and 492 nm) (μM)16-38 hrs. 38-64 hrs. Control 0.433 ± 0.008 0.436 ± 0.060 Example 36 100.397 ± 0.054 0.195 ± 0.017 Example 36 30 0.362 ± 0.026 0.085 ± 0.006Example 36 100 0.290 ± 0.031 0.049 ± 0.006 n = 3; Mean ± StandardDeviation

As is shown in Table 1, the compound of Example 36 inhibits theserum-stimulated proliferation of vascular smooth muscle cellsdose-dependently.

Test Example 2 Inhibitory Effect on Proliferation of Vascular SmoothMuscle Cells (in vitro)

In order to assess the inhibitory effect of a compound of the presentinvention on serum- or PDGF-stimulated proliferation of vascular smoothmuscle cells in vitro, the compound obtained in Example 36 was testedusing A7r5 cells derived from the aorta of a rat (ATCC-CRL-1444).Further, the effect was compared with that of α-tocopherol which wasreported to have an inhibitory effect on proliferation of vascularsmooth muscle cells (see J. Biol. Chem., 266, pp. 6188-6194 (1991)).

Using DMEM medium containing 10% FBS, the cells were placed on 96-wellmicrotiter plates with 5×10³ cells/0.2 ml/well and then incubated. Themedium was exchanged for 2% FBS-containing DMEM medium on the third dayand then for 0.2% FBS-containing DMEM medium on the fourth day from thestart of the incubation. After 48-hour incubation in the 0.2%FBS-containing DMEM medium, the medium was removed, 0.2 ml of DMEMmedium and the test compound were added, and the cells were stimulatedwith serum (2% FBS) or PDGF (PDGF-BB: human origin, available fromBecton Dickinson Labware) (4 ng/ml) to proliferate. The test compoundwas added to the wells in the form of a solution or suspension inethanol in amount of 1 μl well to final compound concentrationsindicated in Table 2 below and the resultant ethanol concentration of0.5% in each well. Eight hours after the stimulation, hydroxyurea wasadded to a final concentration of 1.5 mM to synchronize the cell cyclestrictly. After additional 14-hour incubation, the medium containinghydroxyurea was removed and the cells were washed, and DMEM medium, thetest compound and the proliferation stimulator were added thereto again.Further, [³H]-thymidine (1 μCi/well; 20 μCi/mmol; available from NENResearch) was added. The [³H]-thymidine uptake in 5 hours was measuredas an indication of cell proliferation. The results obtained are shownin Table 2 below.

TABLE 2 Inhibitory Effect on Proliferation of Vascular Smooth MuscleCells (2) Count by Liquid Scintillation Counter Concn. FBS StimulationPDGF Stimulation (μM) (CPM) (CPM) Control 42789 ± 1063 24517 ± 2370α-Tocopherol 30 28058 ± 3912 12493 ± 2978 α-Tocopherol 100 25706 ± 416012159 ± 2362 Example 36 100 14147 ± 1372 7881 ± 356 n = 3; Mean ±standard Deviation

As shown in Table 2 above, the compound of Example 36 inhibits serum- orPDGF-stimulated proliferation of vascular smooth muscle cells. Theinhibitory effect of the compound is higher than that of α-tocopherol.

Test Example 3 Inhibitory Effect on Proliferation of Vascular SmoothMuscle Cells (in vitro)

In order to assess the inhibitory effect of the compounds of the presentinvention on serum-stimulated proliferation of vascular smooth musclecells in vitro, the compounds of Examples 1, 4, 20, 22, 27, 31, 36, 50,52, and 58 were tested using A7r5 cells derived from the aorta of a rat(ATCC-CRL-1444).

Using DMEM medium containing 10% FBS, the cells were placed on 96-wellmicrotiter plates with 5×10³ cells/0.2 ml/ well and then incubated. Themedium was exchanged for 2% FBS-containing DMEM medium on the third dayand then for 0.2% FBS-containing DMEM medium on the fourth day from thestart of the incubation. After 24-hour incubation in the 0.2%FBS-containing DMEM medium, the test compound was added. Each of thetest compounds were added to the wells in the form of a solution orsuspension in ethanol in an amount of 1 μl/well to final compoundconcentrations indicated in Table 3 below and the resultant ethanolconcentration of 0.5% in each well. After additional 24-hour incubation,the medium was removed, and 0.2 ml of DMEM medium and the test compoundwere added thereto again, and the cells were stimulated by serum (2%FBS). Forty-eight hours after the stimulation, the acid phosphataseactivity in the wells was measured by means of a kit (Abacus CellProliferation Kit, available from Clontech Lab. Inc. CA, U.S.A.) andevaluated as an indication of cell proliferation. The results obtainedare shown in Table 3 below.

TABLE 3 Inhibitory Effect on Proliferation of Vascular Smooth MuscleCells (3) Acid Phosphatase Activity Test Concn. Absorbance % ControlCompound (μM) (OD405) (%) Control 0.867 ± 0.089 100 ± 10 Example 1 100.727 ± 0.032 84 ± 4 100 0.288 ± 0.073 33 ± 8 Example 22 10 0.875 ±0.013 101 ± 1  100 0.777 ± 0.062 90 ± 7 Example 31 10 0.756 ± 0.025 87 ±3 100 0.600 ± 0.102  69 ± 12 Example 36 10 0.733 ± 0.060 84 ± 7 1000.539 ± 0.055 62 ± 6 Example 4 10 0.606 ± 0.020 70 ± 2 30 0.062 ± 0.026 7 ± 3 Example 20 10 0.472 ± 0.031 54 ± 4 30 0.105 ± 0.027 12 ± 3Example 27 10 0.553 ± 0.033 64 ± 4 30 0.225 ± 0.064 26 ± 7 Example 50 100.699 ± 0.131  81 ± 15 30 0.146 ± 0.026 17 ± 3 Example 52 10 0.690 ±0.071 80 ± 8 30 0.043 ± 0.008  5 ± 1 Example 58 10 0.417 ± 0.125  48 ±14 30 0.045 ± 0.006  5 ± 1 n = 3, Mean ± Standard Deviation

As is shown in Table 3, all the compounds tested inhibit theserum-stimulated proliferation of vascular smooth muscle cells.

Test Example 4 In Vivo Effect in Intimal Thickening Model (1)

In order to assess the inhibitory effect of the compound of the presentinvention on intimal thickening in vivo, the compound of Example 36 wastested using rabbit balloon injury models.

Cholesterol-fed rabbits in groups of eight (JW: CSK male, 11-week-old)were used to prepare rabbit balloon injury models. Briefly, rabbits werefed with a high cholesterol diet (cholesterol content: 1%) for 2 weeksbefore balloon treatment. The animals were balloon-treated with aballoon catheter (3 French, available from Baxter) under anesthesia topeel the aortic intima 5 times. The animals were orally administeredwith the compound which was suspended in a 1% carboxymethyl cellulose(CMC) solution (200 mg/kg). On the other hand, control animals wereorally administered with a 1% CMC solution without the compound. Theadministrations were performed once a day from 1 week before the balloontreatment until the day before the dissection for evaluation.

After four weeks from the balloon treatment, specimens were taken fromthe aorta, stained with Elastica van Geison, and analyzed under anoptical microscope. The image was processed to measure the thickness andarea of the thickened intima. Averages of the thickness and area wereobtained for each individual animal. The animals which died due to theoperation and those having a serum cholesterol level of 3500 mg/dl orhigher were excluded from the measurement. The number of animals usedfor the evaluation was 5 or 6 per group. The results obtained are shownin Table 4.

TABLE 4 Inhibitory Effect on Intimal Thickening in Balloon Injury Models(1) Compound of Control Example 36 Thickness of  103 ± 12   45 ± 31**Intima (μm) Area of Intima 0.26 ± 0.03 0.11 ± 0.08** (mm²) Means ±Standard Deviation; **P < 0.01

As shown in Table 4, the compound of Example 36 exhibits significantinhibitory effect on intimal thickening in the rabbit balloon injuryintimal thickening models on both parameters of intimal thickness andintimal area.

Test Example 5 In Vivo Effect in Intimal Thickening Model (2)

In order to assess the inhibitory effect of the compound of the presentinvention on intimal thickening in vivo, the compound of Example 36 wastested using rabbit balloon injury models that were different from thoseused in Test Example 4 in week-age. Further, the effect was comparedwith that of Probucol which was reported to have an inhibitory effect onintimal thickening in balloon injury models (see Proc. Natl. Acad. Sci.,U.S.A., 89, pp 11312-11316 (1992)).

Cholesterol-fed rabbits in groups of seven (JW: CSK male, 15-week-old)were used to prepare rabbit balloon injury models. Briefly, rabbits werefed with a high cholesterol diet (cholesterol content: 1%) for 2 weeksbefore balloon treatment. The animals were balloon-treated with aballoon catheter (4 French, available from Baxter) under anesthesia topeel the aortic intima 5 times. The animals were orally administeredwith the compound which was suspended in a 1% CMC solution (200 mg/kg).On the other hand, control animals were orally administered with a 1%CMC solution without the compound. The administrations were performedonce a day from 1 week before the balloon treatment until the day beforethe dissection for evaluation. In order to evaluate the degree ofballoon injury, a balloon-nontreated group (3 animals) was prepared.

After four weeks from the balloon treatment, specimens were taken fromthe aorta, stained with Elastica van Geison, and analyzed under anoptical microscope. The image was processed to measure the thickness andarea of the thickened intima. Averages of the thickness and area wereobtained for each individual animal. The animals which died after theoperation and those having a serum cholesterol level of 3500 mg/dl orhigher were excluded from the measurement. The number of animals usedfor the evaluation was 4 or 5 per group. The results obtained are shownin Table 5.

TABLE 5 Inhibitor Effect on Intimal Thickening in Balloon Injury Models(2) Compound of Balloon- Control Probucol Example 36 nontreatedThickness 119 ± 19 92 ± 17 76 ± 16** 46 ± 20** of Intima (μm) Area of1.32 ± 0.22 0.97 ± 0.28 0.81 ± 0.22* 0.45 ± 0.21** Intima (μ2) Mean ±Standard Deviation; *: P<0.05; **: P<0.01

As can be seen from the results in Table 5, the compound of Example 36exhibits significant inhibitory effect on intimal thickening in therabbit balloon injury induced intimal thickening models on bothparameters of intimal thickness and intimal area. The effect is higherthan that of Probucol.

INDUSTRIAL APPLICABILITY

The vascular intimal thickening inhibitory agent according to thepresent invention which comprises a 2,6-di-t-butylphenol derivative asan active ingredient shows a potent inhibitory action on cellproliferation caused by serum or PDGF in cultured vascular smooth musclecells as well as a potent inhibitory effect on intimal thickening inballoon injury intimal thickening models. Therefore, the agent is usefulfor the treatment and prevention of restenosis due to vascular intimalthickening following PTCA.

What is claimed is:
 1. A method for inhibiting proliferation of vascularsmooth muscle cells, or for inhibiting thickening or vascular stenosisfollowing percutaneous transluminal coronary angioplasty, comprisingadministering to a patient in need of said therapy an effective amountfor said therapy of a compound of formula (1)

wherein X is an oxygen atom; R₁ is a hydrogen atom or an acyl group; R₂is a hydrogen atom, a lower alkyl group or a lower alkenyl group; R₃ andR₄ are taken together to form a 5-membered ring; R₅ is a substituted orunsubstituted alkyl group having 1 carbon atom; and R₆ is a substitutedor unsubstituted alkyl group having 1 to 20 carbon atoms, a substitutedor unsubstituted alkenyl group having 2 to 20 carbon atoms, asubstituted or unsubstituted alkynyl group having 2 to 20 carbon atoms,or a substituted or unsubstituted aryl group; provided that R₆ is nilwhen R₃ and R₄ are taken together to form a benzofuran ring.
 2. Themethod of claim 1, wherein R₁ and R₂ are both a hydrogen atom.
 3. Themethod of claim 2, wherein R₆ is a substituted or unsubstituted alkylgroup having 1 to 20 carbon atoms or a substituted or unsubstitutedalkenyl group having 2 to 20 carbon atoms.